Promoters regulate gene transcription and provide sophisticated controlling mechanisms for gene expression. Based on their functionality, promoters can be generally grouped into tissue-specific promoters, developmentally regulated promoters, constitutive promoters and inducible promoters. Tissue-specific promoters become functionally active only in a particular type of cell, tissue or organ, while developmentally regulated promoters mainly function at a certain stage of plant development. Constitutive promoters, on the other hand, are active in all types of tissues and at all stages of plant development, providing a relatively constant level of gene expression. Lastly, inducible promoters, increase activity greatly when they come in contact with associated chemical or physical signals. Thus far, numerous promoters with a wide range of functionalities have been isolated from organisms ranging from viruses to plants to mammals. These promoters are potentially important for the successful application of genetic engineering techniques to crop plants in efforts to introduce resistance characteristics to biotic and abiotic stresses, to improve crop yield and quality, and to create novel traits with added value.
Currently, the majority of promoters being successfully utilized in plant genetic engineering are mainly derived from a few plant viruses, including cauliflower mosaic virus (CaMV) and cassava vein mosaic virus (CsVMV). These viral promoters tend to support strong and often constitutive transgene expression in plant cells (Li et al., 2001). Hence, they are normally used for expressing selectable marker genes to facilitate the selection and identification of transgenic plants. In addition, due to their unusually stable and high-level of activity, these promoters are often used to drive expression of transgenes such as those conferring resistance to disease pathogens, insects and herbicides (D'Halluin et al., 1993; Lonstaff et al., 1993; Perlak et al., 1990; Ger et al., 2002). However, viral promoters generally lack the capability to promote tissue-specific expression and some show unpredictable expression patterns in different host plants (Li et al., 2001; Kloti et al., 2002). In addition to viral promoters, several promoters isolated from plants have also been successfully used in genetic engineering programs (An et al., 1996; Dharmapuri et al., 2002). These plant-derived promoters are capable of supporting tissue-specific and developmentally-regulated transgene expression, but more often display unexpected changes in promoter activity and expression patterns in heterologous host plants (Li et al., 2001). The use of plant-derived promoters for efficient transgene expression has been somewhat under-exploited. More needs to be done in the discovery, isolation and characterization of novel plant-derived promoters from different plant species and in the utilization of these promoters in order to diversify and enhance our ability to achieve effective transgene expression in transgenic plants.
Promoters are a key component in the application of genetic engineering techniques aimed at improving the survivability and value of crop plants. In spite of tremendous efforts made to isolate and characterize promoters from various sources, the number of promoters capable of providing a strong and tissue-specific activity in a particular host plant is still limited mainly due to the sophisticated mechanisms employed by different promoters to control gene expression (Li et al., 2001). Thus, there is a great need for discovery and exploration of new promoters in order to diversify our approach to supporting transgene expression in various plant species for different purposes.
2S albumins are a group of small proteins (1.7 to 2.2S) synthesized and stored in plant seeds of a wide range of dicotyledonary species and are well known for their conserved pattern of placement of cysteine residues that are involved in disulphide bond formation (Shewry et al., 1995; Rico et al., 1996). These proteins vary greatly in amino acid composition but often contain a high percentage of nitrogen- and sulfur-rich amino acid residues consistent with significant nutritional functions of storage proteins (Boutilier et al., 1999). Due to their important role as a nutritional supply source for human food and animal feeds, 2S albumins and associated genes have been isolated from a number of plant species and characterized in great detail to facilitate endeavors in protein manipulation and improvement (Raynal et al., 1991; Shewry et al., 1995; Rico et al., 1996; Cahtthai and Misra 1998; Muntz 1998; Scarafoni et al., 2001; Galili and Hofgen 2002). Several glutamine (Q)-rich 2S albumins have also been shown to possess potent antimycotic and antibacterial activities. These proteins provide useful characteristics for the development of novel disease resistance in crop plants using transgenic strategies (Terras et al., 1993; Barciszewski et al., 2000; Koo et al., 2002).
The production and accumulation of 2S albumins during seed development are strictly controlled by their gene promoters. Over the years, extensive molecular analysis of 2S albumin promoters from different plant species revealed that several unique DNA motifs within these promoters interact directly with well-characterized transcriptional regulatory proteins leading to spatial and temporal activation of gene transcription (Dasgupta et al., 1993; Stalberg et al., 1993; Conceicao and Krebbers 1994; Rockel et al., 1997; Vincentz et al., 1997). Knowledge gained from these studies greatly facilitated efforts to improve yield, quality and nutritional value of seed storage proteins by using genetic engineering techniques (Molvig et al., 1997; Muntz et al., 1998; De Lumen et al., 1999; Scarafoni et al., 2001; Jaeger et al., 2002).
A cDNA clone from a grape berry library of cv. Pinor Noir was published previously in the form of an expressed sequence tag (EST) (Ageorges 2000). This partial cDNA was shown to have sequence homology to a gene encoding a 2S albumin seed storage protein precursor from Juglans regia (U66866) and thus was proposed to belong to a 2S albumin-like gene in grape. Although the isolation and utilization of 2S albumin genes and promoters from other species has been disclosed in the art, none have sequence homology to the gene and promoter of the subject invention (Arntzen et al., 2002; Vandekerckhove et al., 1997; De Clercq et al., 1996; Vandekerckhove et al., 1996).